The condensing action of cholesterol has been compared with that of a structural isomer having its hydroxyl group located at the C-25 position (i.e., 25-OH'), that is, an isomer favoring an "upside down" orientation in lipid membranes. Surface pressure-area isotherms of mixed monolayers made from 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/cholesterol and DMPC/25-OH' have established that 25-OH' has a weaker condensing effect than cholesterol. Nearest-neighbor recognition measurements in liposomes made from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) have also shown that 25-OH' has a weaker condensing effect in the physiologically relevant fluid bilayer state. These findings provide support for surface occupancy playing a role in the condensing action of cholesterol.